加州鲈肌肉生长抑制素(MSTN)cDNA的克隆和序列分析

Molecular cloning and sequence analysis of Myostatin in largemouth bass (Micropterus salmoides)

肌肉生长抑制素是抑制肌肉生长和发育的生长调控因子。对运用RT—PCR和RACE技术从加州鲈成鱼肌肉总RNA中扩增得到的MSTNcDNA全序列进行了序列分析。结果表明，加州鲈MSTNcDNA全长为1626bp，其开放阅读框为1134bp，共编码377个氨基酸，前面的22个氨基酸为信号肽，中间有四个氨基酸（RARR）为蛋白水解加工位点；该基因总共有13个半胱氨酸残基，后面9个在蛋白水解加工位点之后的c端生物活性区，与其它脊椎动物比较，它们的位置完全一致，对该基因的结构和功能非常重要。与GenBank中已知的条纹狼鲈、金鲷、斑马鱼、虹鳟、斑点叉尾鲴、人、猪、鸡、鸽MSTN的ORF相比较，核苷酸序列同源性为63％-94．4％，氨基酸同源性为61．4％-96％，特别是在C端生物活性区氨基酸同源性为88．1％-100％，高度的保守性反映了该基因受到了高度的进化限制以及功能的重要性。加州鲈MSTN基因的克隆为研究该基因打靶和鱼类肌肉发育调控机理奠定了基础。

Myostatin, as a negative regulator of myogenesis, inhibits myoblast proliferation and differentiation. Total RNA was isolated from muscle of adult largemouth bass to clone the MSTN cDNA by using rapid cDNA end amplification and reverse transcription polymerase chain reaction. The sequence was analyzed, the results showed that the full-length of largemouth bass MSTN was 1626 bp with an open read frame（ORF） of 1 134 bp enconding 377 amino acids; The predicted amino acid sequence contained a putative signal peptide of 22amino acids, a conserved proteolytic processing site （RARR） and bioactive carboxy terminal region including 9 cysteine residues. Comparing largemouth bass MSTN with those of striped bass, gilthead seabream, mozambique tilapia, rainbow trout, zebrafish, pigeon, mouse and human, the results revealed that the nucleotide homology was between 63% -94.4% and amino acid homology between 61.4% -96%. Of particular interest, bioactive carboxy terminal region, which lay downstream of each processing site, was considerably conserved. They were 88. 1% - 100% identical within these genus. High level of sequence conservation among these known MSTN orthologues suggested that it was highly restricted in the process of evolution and its function was conservative. Cloning of largemouth bass MSTN is meaningful for researching gene targeting and the mechanism of fish＇s muscle development and growth.